Low dose combination CDA substrate drug/cedazuridine with extended administration

ABSTRACT

This invention relates to methods and compositions for administering an effective amount of a CDA substrate drug and an effective amount of cedazuridine. In particular, the invention relates to methods for treating cancer, inhibiting degradation of a CDA substrate drug, and reducing DNA methylation in a subject in need thereof comprising administering an effective amount of a CDA substrate drug and an effective amount of cedazuridine.

STATEMENT OF PRIORITY

This application claims the benefit, under 35 U.S.C. § 119(e), of U.S.Provisional Application No. 62/733,277, filed on Sep. 19, 2018, theentire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to methods and compositions for administering aneffective amount of a CDA substrate drug and an effective amount ofcedazuridine. In particular, the invention relates to methods fortreating cancer, inhibiting degradation of a CDA substrate drug, andreducing DNA methylation in a subject in need thereof comprisingadministering an effective amount of a CDA substrate drug and aneffective amount of cedazuridine.

BACKGROUND OF THE INVENTION

The enzymes adenosine deaminase (ADA, EC 3.5.4.4) and cytidine deaminase(CDA, EC 3.5.4.5) function to deaminate natural aminopurine andaminopyrimidine nucleosides, respectively, in human and other organisms.They may also convert active nucleoside-based drugs into inactivemetabolites. For example, the purine nucleoside drug arabinosyladenine(fludarabine, ara-A) is deaminated by ADA; the resulting compound, withthe parent amino group replaced with hydroxyl, is inactive as anantitumor agent compared to the parent compound. Similarly, theantileukemia drug arabinosylcytosine (cytarabine, also known as ara-C)is metabolically degraded by CDA into inactive arabinosyluracil.

CDA is a component of the pyrimidine salvage pathway. It convertscytidine and deoxycytidine to uridine and deoxyuridine, respectively, byhydrolytic deamination (Arch. Biochem. Biophys. 1991, 290, 285-292;Methods Enzymol. 1978, 51, 401-407; Biochem. J. 1967, 104, 7P). It alsodeaminates a number of synthetic cytosine analogs which are clinicallyuseful drugs, such as ara-C (Cancer Chemother. Pharmacol. 1998, 42,373-378; Cancer Res. 1989, 49, 3015-3019; Antiviral Chem. Chemother.1990, 1, 255-262). Conversion of the cytosine compounds to the uridinederivatives usually confers loss of therapeutic activity or addition ofside-effects. It has also been shown that cancers that acquireresistance to cytosine analog drugs often overexpress CDA (Leuk Res.1990, 14, 751-754). Leukemic cells expressing a high level of CDA canmanifest resistance to cytosine antimetabolites and thereby limit theantineoplastic activity of such therapeutics (Biochem. Pharmacol. 1993,45, 1857-1861).

The present invention overcomes shortcomings in the art by providingmethods for treating cancer, inhibiting degradation of a CDA substratedrug, and/or reducing DNA methylation in a subject in need thereofcomprising administering an effective amount of a CDA substrate drug andan effective amount of cedazuridine.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the development of low dose(LD) administration schedules of combination decitabine/cedazuridinethat result in improved epigenctic effects and decreasedmyelosuppressive effects in cancer patients.

Thus, one aspect of the invention relates to methods for treating cancerin a subject in need thereof, comprising administering to the subject:(i) an effective amount of a CDA substrate drug (e.g., decitabine); and(ii) an effective amount of cedazuridine, thereby treating cancer in thesubject.

An additional aspect of the invention relates to methods for inhibitingdegradation of a CDA substrate drug (e.g., decitabine, e.g.,5-azacytidine) in a subject in need thereof, comprising administering tothe subject: (i) an effective amount of a CDA substrate drug (e.g.,decitabine, e.g., 5-azacytidine); and (ii) an effective amount ofcedazuridine, thereby inhibiting degradation of the CDA substrate drugin the subject.

A further aspect of the invention relates to methods of reducing DNAmethylation (e.g., LINE-1 methylation) in a subject in need thereof,comprising administering to the subject: (i) an effective amount of aCDA substrate drug; and (ii) an effective amount of cedazuridine,thereby reducing DNA methylation in the subject.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows neutrophil dynamics in three patients after guadecitabinetreatment of AML patients (“responders”) using daily dosing on 5consecutive days every 28 days. Fitting results, observed vs. predicted.

FIG. 2 shows key differential equations used in the quantitative systemspharmacology model as described in Example 1.

FIGS. 3A-3B show simulations of decitabine in plasma (FIG. 3A) andneutrophil dynamics (FIG. 3B) during treatment of virtual MDS patientswith varying regimens of ASTX727. Regimens simulated included: (black)35 mg decitabine and 100 mg cedazuridine (“ 35/100 mg ASTX727”) dailyfor 3 consecutive days every 28-day cycle; (blue) 20 mg decitabine and100 mg cedazuridine (“ 20/100 mg ASTX727”) daily for 3 consecutive daysduring two weeks (6 days total) every 28-day cycle; (red) 10 mgdecitabine and 100 mg cedazuridine (“ 10/100 mg ASTX727”) daily for 3consecutive days during three weeks (9 days total) every 28-day cycle;and (green) 5 mg decitabine and 100 mg cedazuridine (“ 5/100 mgASTX727”) daily for 5 consecutive days during two weeks (10 days total)every 28-day cycle.

FIG. 4 shows decitabine concentrations detectable in patient plasma atindicated time points. C1D1 refers to cycle 1, day 1; C1D5 refers tocycle 1, day 5. Data is cumulative of patients in the 5 mg cohort (toppanel) and 10 mg cohort (bottom panel) identified in Table 1.

FIG. 5 shows % demethylation of LINE-1 achieved in the cohorts ofExample 3. Groups identified in the figure legend correspond to thecohorts as listed in Table 6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, nucleotide sequences,amino acid sequences and other references mentioned herein areincorporated by reference in their entirety.

Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specifiedamount.

As used herein, the transitional phrase “consisting essentially of” isto be interpreted as encompassing the recited materials or steps andthose that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

“Effective amount” refers to the amount required to produce a desiredeffect (e.g., enhancing the half-life, bioavailability or efficacy of aCDA substrate drug, treating cancer in a subject, reducing DNAmethylation in a subject, inhibiting cytidine deaminase in a subject, orinhibiting degradation of a CDA substrate drug by cytidine deaminase).

“Half-life” refers to the period of time required for the concentrationor amount of a compound in a subject to be reduced to exactly one-halfof a given concentration or amount.

“Pharmaceutically acceptable” refers to those properties and/orsubstances that are acceptable to the patient from a pharmacologicaland/or toxicological point of view, and/or to the manufacturingpharmaceutical chemist from a physical and/or chemical point of viewregarding composition, formulation, stability, patient acceptance,bioavailability and compatibility with other ingredients.

“Pharmaceutically acceptable excipient” can mean any substance, notitself a therapeutic agent, used as a carrier, diluent, adjuvant,binder, and/or vehicle for delivery of a therapeutic agent to a subject,or added to a pharmaceutical composition to improve its handling orstorage properties or to permit or facilitate formation of a compound orcomposition into a unit dosage form for administration. Pharmaceuticallyacceptable excipients are well known in the pharmaceutical arts and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa. (e.g., 20th Ed., 2000), and Handbook ofPharmaceutical Excipients, American Pharmaceutical Association,Washington, D.C., (e.g., 1st, 2nd and 3rd Eds., 1986, 1994 and 2000,respectively). As will be known to those skilled in the art, excipientsmay provide a variety of functions and may be described as wettingagents, buffering agents, suspending agents, lubricating agents,emulsifiers, disintegrants, absorbents, preservatives, surfactants,colorants, flavorants, and sweeteners. Examples of pharmaceuticallyacceptable excipients include without limitation: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, cellulose acetate,hydroxypropylmethylcellulose, and hydroxypropylcellulose; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such ascocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

“Pharmaceutically acceptable salt” refers to an acid or base salt of acompound of the invention, which salt possesses the desiredpharmacological activity and is neither biologically nor otherwiseundesirable. The salt can be formed with acids that include withoutlimitation acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride hydrobromide,hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,thiocyanate, tosylate and undecanoate. Examples of a base salt includewithout limitation ammonium salts, alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such as arginineand lysine. In some embodiments, the basic nitrogen-containing groupscan be quarternized with agents including lower alkyl halides such asmethyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkylsulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aralkyl halides such as phenethyl bromides.

“Unit dosage form” refers to a physically discrete unit suitable as aunitary dosage for human or other animal subjects. Each unit dosage formmay contain a predetermined amount of an active substance (e.g.,compound or composition of the invention, CDA substrate drug and/orother therapeutic agent) calculated to produce a desired effect.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. For example, an alkyl that is “optionally substituted”encompasses both an alkyl that is unsubstituted and an alkyl that issubstituted.

The term “enhance” or “increase” refers to an increase in the specifiedparameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, fifteen-fold, etc.

The term “inhibit” or “reduce” or grammatical variations thereof as usedherein refers to a decrease or diminishment in the specified level oractivity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%,95% or more. In particular embodiments, the inhibition or reductionresults in little or essentially no detectable activity (at most, aninsignificant amount, e.g., less than about 10% or even 5%).

“Subject” refers to a cell or tissue, in vitro or in vivo, an animal ora human. An animal or human subject may also be referred to as a“patient.”

“Animal” refers to a living organism having sensation and the power ofvoluntary movement, and which requires for its existence oxygen andorganic food.

“Mammal” refers to a warm-blooded vertebrate animal with hair or fur.Examples include without limitation members of the human, equine,porcine, bovine, murine, canine or feline species.

By the term “treat,” “treating,” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved. “Treating” in reference to a disease,disorder or condition may refer to: (i) inhibiting a disease, disorderor condition, e.g., arresting its development; and/or (ii) relieving adisease, disorder or condition, e.g., causing regression of the clinicalsymptoms.

“Preventing” in reference to a disease, disorder or condition refers topreventing a disease, disorder or condition, e.g., causing the clinicalsymptoms of the disease, disorder or condition not to develop. As usedherein, the term “prevent,” “prevents,” or “prevention” (and grammaticalequivalents thereof) may also refer to a delay in the onset of a diseaseor disorder or the lessening of symptoms upon onset of the disease ordisorder. The terms are not meant to imply complete abolition of diseaseand encompass any type of prophylactic treatment that reduces theincidence of the condition or delays the onset and/or progression of thecondition.

The term “administering” or “administration” of a compound and/orcomposition of the present invention to a subject includes any route ofintroducing or delivering to a subject a compound to perform itsintended function. Administration can be carried out by any suitableroute, including orally, intranasally, parenterally (intravenously,intramuscularly, intraperitoneally, intracisternally, intrathecally,intraventricularly, or subcutaneously), or topically. Administrationincludes self-administration and the administration by another.

“Cancer” refers to an abnormal growth of cells which tend to proliferatein an uncontrolled way and, in some cases, to metastasize (spread).Specific cancers types include without limitation the cancers identifiedin Publication No. US 2006/0014949 and the following: cardiac: sarcoma(e.g., such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcomaand the like), myxoma, rhabdomyoma, fibroma, lipoma and teratomas; lung:bronchogenic carcinoma (e.g., such as squamous cell, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma and the like),alveolar (e.g., such as bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;gastrointestinal: esophagus (e.g., such as squamous cell carcinoma,adenocarcinoma, leiomyosarcoma, lymphoma and the like), stomach (e.g.,such as carcinoma, lymphoma, leiomyosarcoma and the like), pancreas(e.g., such as ductal adenocarcinoma, insulinoma, glucagonoma,gastrinoma, carcinoid tumors, vipoma and the like), small bowel (e.g.,such as adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma, and the like),large bowel (e.g., such as adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma and the like); genitourinary tract: kidney(e.g., such as adenocarcinoma, Wilm's tumor nephroblastoma, lymphoma,leukemia, and the like), bladder and urethra (e.g., such as squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma and thelike), prostate (e.g., such as adenocarcinoma, sarcoma), testis (e.g.,such as seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma and the like); liver: hepatoma(e.g., hepatocellular carcinoma and the like), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone:osteogenic sarcoma (e.g., such as osteosarcoma and the like),fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing'ssarcoma, malignant lymphoma (e.g., such as reticulum cell sarcoma),multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma(e.g., such as osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; nervous system: skull (e.g., such as osteoma, hemangioma,granuloma, xanthoma, osteitis deformans and the like), meninges (e.g.,such as meningioma, meningiosarcoma, gliomatosis and the like), brain(e.g., such as astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors and the like), spinal cord(e.g., such as neurofibroma, meningioma, glioma, sarcoma and the like);gynecological: uterus (e.g., such as endometrial carcinoma and thelike), cervix (e.g., such as cervical carcinoma, pre-tumor cervicaldysplasia and the like), ovaries (e.g., such as ovarian carcinoma[serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma, and the like), vulva (e.g., such assquamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma,fibrosarcoma, melanoma and the like), vagina (e.g., such as clear cellcarcinoma, squamous cell carcinoma, botryoid sarcoma (embryonalrhabdomyosarcoma], fallopian tubes (carcinoma) and the like);hematologic: blood (e.g., such as myeloid leukemia [acute and chronic],acute lymphoblastic leukemia, chronic lymphocytic leukemia,myeloproliferative diseases, multiple myeloma, myelodysplastic syndromeand the like), Hodgkin's disease, non-Hodgkin's lymphoma; skin:malignant melanoma, basal cell carcinoma, squamous cell carcinoma,Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis and the like; and adrenal glands:neuroblastoma.

Methods

The present invention is based, in part, on the development of low dose(LD) administration schedules of combination CDA substratedrugs/cedazuridine that result in improved epigenetic effects anddecreased myelosuppressive effects in cancer patients. “Low dose” asused herein refers to lower cumulative dose amount per cycle oftreatment of a CDA substrate drug (e.g., decitabine, e.g.,5-azacytidine) and/or cedazuridine that are lower than the standardapproved dose amounts for these compounds (e.g., when administeredalone), either through reduced per-dose amounts and or through modifiedschedules (e.g., number of days of administering per treatment cycle;e.g., spacing of days of administering per treatment cycle). While notwishing to be bound to theory, the inventors of the present inventionhave found that low dose administration schedules of decitabine andcedazuridine may provide equivalent or improved epigenetic effects(e.g., LINE-1 demethylation, e.g., % F cell expansion) and/or decreasedmyelosuppressive effects (e.g., neutropenia) in IPSS-low andIPSS-intermediate-1 MDS cancer patients.

Thus, the present invention provides methods of administering (i) aneffective amount of a CDA substrate drug, and (ii) an effective amountof cedazuridine.

As used herein, the term “CDA substrate drug” refers to a drug that canbe deaminated by the enzyme cytidine deaminase (CDA). Nonlimitingexamples of a CDA substrate include cytidine analogs, such asdecitabine, 5-azacytidine, gemcitabine, ara-C (also known ascytarabine), troxacitabine, tezacitabine, 5′-fluoro-2′-deoxycytidine,and cytochlor.

Thus, in some embodiments, the present invention provides a method fortreating cancer in subject in need thereof, comprising administering tothe subject: (i) an effective amount of a CDA substrate drug; and (ii)an effective amount of cedazuridine, thereby treating cancer in thesubject.

Cedazuridine ((4R)-2′-deoxy-2′,2′-difluoro-3,4,5,6-tetrahydrouridine;also known as E7727) is a recently developed CDA inhibitor. Cedazuridinein combination with decitabine is in development as a single oralcomposition referred to as ASTX727. Cedazuridine and methods of makingand/or using thereof are further disclosed in U.S. Pat. No. 8,268,800,the contents of which are incorporated by reference herein in theirentirety.

The CDA substrate drug of the present invention may be any drug that canbe deaminated by CDA. In some embodiments, the CDA substrate drug may be5-azacytidine. In some embodiments, the CDA substrate drug may bedecitabine.

Decitabine (5-aza-2′-deoxycytidine) is an antineoplastic agent andhypomethylating agent (HMA) for the treatment of myelodysplasticsyndrome (MDS), with potential utility for the treatment of AML and CMLas well. Like other cytidine-based drugs, its oral bioavailability andefficacy are limited by deactivation by CDA. Tetrahydrouridine (THU), aninhibitor of CDA, has been shown to improve the potency of decitabine ina sickle cell disease model in baboons (Am. J. Hematol. 1985, 18,283-288). In addition, another known CDA inhibitor, zebularine, has beenshown to enhance the efficacy of decitabine in mice with L1210 leukemia(Anticancer Drugs 2005, 16, 301-308).

The cancer treated by the methods of the invention may be any canceragainst which the CDA substrate drug is known or later discovered to beeffective. In some embodiments, the cancer is selected fromhematological cancers and solid cancers. The hematological cancer maybe, for example, MDS, leukemia (e.g., ALL, AML, CML, or CMML), orlymphoma (e.g. Hodgkin's Lymphoma, Non-hodgkin lymphoma, or T-celllymphoma). Examples of solid cancers include, but are not limited to,pancreatic cancer, ovarian cancer, peritoneal cancer, non small celllung cancer, breast cancer, neuroectodermal tumors, and sarcomas.

In some embodiments, the present invention provides a method fortreating cancer, wherein the cancer is MDS. MDS is a group of cancerdisorders with shared improper maturation of bone marrow derived bloodcells, and symptoms can vary. The International Prognostic ScoringSystem (IPSS) scores MDS severity into several risk groups based onpatient hemoglobin levels, absolute neutrophil count (ANC), plateletcount, and percent bone marrow blasts. Risk groups include “low”,“intermediate-1”, “intermediate-2”, and “high”. Other risk group scoringmethodologies exist such as the Revised IPSS (IPSS-R) and the WHOclassification-based Prognostic Scoring System (WPSS), as described inthe NCCN Guidelines for Patients, Myelodysplastic Syndromes, 2018.Methods of the present invention may be used to treat cancer in asubject (e.g., a human patient) diagnosed with any risk group of MDSbased on any scoring methodology. In some embodiments, a subject in needof the present invention may include a subject diagnosed with lower riskMDS (e.g., IPSS low and/or intermediate-1).

In some embodiments, the present invention provides a method forinhibiting degradation of a CDA substrate drug (e.g., decitabine, e.g.,5-azacytidine) in a subject in need thereof, comprising administering tothe subject: (i) an effective amount of a CDA substrate drug; and (ii)an effective amount of cedazuridine, thereby inhibiting degradation ofthe CDA substrate drug in the subject.

In some embodiments, the present invention provides a method of reducingDNA methylation in a subject in need thereof, comprising administeringto the subject: (i) an effective amount of a CDA substrate drug; and(ii) an effective amount of cedazuridine, thereby reducing DNAmethylation in the subject (e.g., thereby enabling reduction in DNAmethylation by the CDA substrate drug). In some embodiments, theadministering reduces DNA methylation of LINE-1. LINE-1 is a longinterspersed nuclear element found in human DNA (e.g., the subject)known in the art, and its methylation levels can be measured usingstandard techniques in the art to determine genetic effects ofhypomethylating agents such as CDA substrate drugs (e.g., decitabine).

The administering to a subject in need thereof of (i) an effective mountof a CDA substrate drug, and (ii) an effective amount of cedazuridinehas been shown by the inventors of the present invention to providemultiple beneficial responses to the subject. For example, in someembodiments, the administering reduces DNA methylation in the subject byat least 5% (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% ormore or any value or range therein) as compared to a controlmeasurement, e.g., as compared to DNA methylation in the subject priorto the administering (e.g., subject “baseline” DNA methylation). DNAmethylation in the subject may be quantitatively and/or qualitativelyevaluated by any standard technique in the art, e.g., as measured by amarker of relative global methylation as compared to a control, e.g., asmeasured by LINE-1 methylation as compared to a control. For example, insome embodiments, the administering reduces LINE-1 methylation in thesubject by at least 5% (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15% or more) as compared to a control measurement, e.g., ascompared to LINE-1 methylation in the subject prior to the administering(e.g., e.g., subject baseline LINE-1 methylation). For example, in someembodiments, the administering may reduce LINE-1 methylation in thesubject by at least 5%, at least 8%, at least 10% or at least 15% ormore. In some embodiments, the administering may reduce LINE-1methylation in the subject by about 5% to about 20%, about 6% to about15%, or by about 8% to about 10%.

In some embodiments, the administering may reduce absolute neutrophilcount (ANC) in the subject to less than 0.5×10⁹ cells/L of blood for nomore than two weeks (e.g., no more than 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 consecutive days or any value or range therein)following a 28-day cycle. In some embodiments, the administering reducesabsolute neutrophil count (ANC) in the subject to less than 0.5×10⁹cells/L of blood for no more than two weeks (e.g., more than two, three,four, five, six weeks etc.) during treatment (e.g., between multiple,repeated 28-day cycles).

In some embodiments, a time period of 0 to 31 days or more (e.g., 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more) may pass betweenmultiple 28-day treatment cycles of the present invention. The timeperiod of no treatment may be desirable to allow a subject (e.g., ahuman patient) of the present invention to have adequate health tocontinue treatment. The time period between treatment cycles can bedetermined by a physician using standard techniques in the art and maybe determined individually on a per-subject basis, for example, as basedon adequate blood count, e.g., adequate lack of neutropenia (e.g.,absolute neutrophil count (ANC) in the subject of at least or greaterthan 0.5×10⁹ cells/L), and may be adjusted over the course of treatmentbased on the judgement of the administering physician. In someembodiments, the time period between treatment cycles may be minimal,e.g., no time period, e.g., immediately starting on the next 28-day timeperiod. In some embodiments, the time period between treatment cyclesmay be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more.

In some embodiments, the administering expands hemoglobin F-expressingcells (i.e., F cells) by at least 5% (e.g., at least 5, 6, 7, 8, 9, 10,15, 20, 25, or 30% or more), optionally as measured by % Fcells/erythrocytes per sample (e.g., in a patient blood sample) ascompared to a “baseline” control % F cells/erythrocytes (e.g., ascompared to % F cells/erythrocytes of the patient prior to treatment,e.g., as compared to the average % F cells/erythrocytes of a patientpopulation not undergoing treatment (e.g., a healthy patientpopulation)). For example, in some embodiments, the administering mayexpand % F cells in the subject by at least 5%, at least 8%, at least10%, at least 15%, or at least 23% or more as compared to a baselinecontrol. In some embodiments, the administering may expand % F cells inthe subject by about 5% to about 30%, about 6% to about 24%, or by about8% to about 20% as compared to a baseline control.

In some embodiments, the administering expands F cells to a total amountof at least 10% to at least 30% or more of total erythrocytes (e.g., atleast 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, or 30% or more F cells/erythrocytes or any value or rangetherein) per sample (e.g., in a patient blood sample). For example, insome embodiments, the administering may expand F cells to a total amountof at least 15%, at least 20%, at least 23%, at least 35% or more oftotal erythrocytes in a sample. In some embodiments, the administeringmay expand F cells to a total amount of about 15% to about 30%, about18% to about 25%, or about 15% to about 35%, of total erythrocytes in asample.

In some embodiments of the methods of the present invention, the subjectmay be a mammal. In some embodiments of the methods of the presentinvention, the subject may be a human.

Administration of the compounds or composition of the invention may bevia any accepted mode known to one skilled in the art, for example,orally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, intraoccularly, intrapulmonarily, or via animplanted reservoir. The term “parenterally” includes without limitationsubcutaneously, intravenously, intramuscularly, intraperitoneally,intrathecally, intraventricularly, intrasternally, intracranially,intratumorally, by injection into a blood vessel feeding a tumor, byintraosseous injection and by infusion techniques. In some embodiments,the CDA substrate drug and cedazuridine may be administeredintravenously, orally, and/or subcutaneously.

The administering of (i) and (ii) in the methods of the presentinvention may be performed prior to, at substantially the same timewith, or after one another. In some embodiments of the methods of thepresent invention, the administering of (i) is concurrent with theadministering of (ii). The term “concurrent” as used herein encompasseswherein the administering of (i) may be immediately followed by theadministering of (ii), and/or wherein the administering of (ii) may beimmediately followed by the administering of (i) (e.g., at substantiallythe same time, e.g., less than 30 minutes, e.g., less than 30, 25, 20,15, 10, 5 or less minutes apart). The term “concurrent” as used hereinalso encompasses wherein the administering of (i) and (ii) is performedtogether, e.g., concomitant, e.g., in a single (e.g., combined)composition.

In some embodiments, the administering of (i) may be performed prior tothe administering of (ii) (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 hrs). In some embodiments, the administering of (ii) isperformed prior to the administering of (i) (e.g., within 1, 2, 3, 4, 5,6, 7, 8, 8, 9, 10, 11, or 12 hrs). In some preferred embodiments, theadministering of (i) may be performed prior to the administering of (ii)within, e.g., 1, 2, or 3 hrs. In some preferred embodiments, theadministering of (ii) may be performed prior to the administering of (i)within, e.g., 1, 2, or 3 hrs.

Any administration regimen well known to those skilled in the art forregulating the timing and sequence of drug delivery can be used andrepeated as necessary to effect treatment in the methods of theinvention. For example, the compounds or composition of the inventionmay be administered 1, 2, 3 or 4 times daily, by a single dose, multiplediscrete doses or continuous infusion. In some embodiments, theadministering of (i) and (ii) may be performed about 1 day to about 28days per 28-day cycle (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28days per 28-day cycle). In some embodiments, the administering of (i)and (ii) may be performed on consecutive days per 28-day cycle. Forexample, (i) and (ii) may be administered on any 2 consecutive days. Insome embodiments, (i) and (ii) may be administered on any 3 consecutivedays (e.g., on a Monday, Tuesday, and a Wednesday “MTW”; on a Tuesday,Wednesday, and a Thursday “TWTh”; on a Wednesday, Thursday, and a Friday“WThF”; on a Thursday, Friday, and Saturday “ThFS”; on a Friday,Saturday, and a Sunday “FSS”; on a Saturday, Sunday, and a Monday “SSM”;and/or on a Sunday, Monday, and a Tuesday “SMT”, etc.). In someembodiments, the administering of (i) and (ii) may be performed on 5consecutive days (e.g., MTWThF or any other combination of 5 consecutivedays), on 7 consecutive days (MTWThFSS or any other combination of 7consecutive days), on 14 consecutive days (e.g., two consecutive weeks);21 consecutive days (e.g., three consecutive weeks), and/or on 28consecutive days (e.g., four consecutive weeks) per 28-day cycle. Theconsecutive day treatments may be repeated one or more times per 28-daycycle, e.g., every week, every other week.

In some embodiments, the administering of (i) and (ii) may be performedon non-consecutive days per 28-day cycle, e.g., for 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, or 27 or more non-consecutive days. Non-consecutive days maycomprise a schedule of every-other day, (e.g., MWF), every two days,every three days, every four days, every five days, every six days,every seven days, etc. in a 28-day cycle. Non-consecutive days maycomprise administering for a number of consecutive days (e.g., “on”),followed by a number of days without administering (e.g., “off”),followed by administering for a number of consecutive days (e.g., “on”),etc., within a 28-day cycle. For example, in some embodiments, theadministering may be performed on 2 non-consecutive days (e.g., everyMonday and Friday or any other combination of 2 non-consecutive days).In some embodiments, the administering may be performed on 3non-consecutive days (e.g., MWF or any other combination of 3non-consecutive days). The non-consecutive day treatments may berepeated one or more times per 28-day cycle, e.g., every week, everyother week.

In some embodiments, the administering may be performed, e.g., for 10total days per 28-day cycle, wherein the 10 days of administeringcomprise 5 consecutive days administering (e.g., 5 days “on”), followedby 2 consecutive days without administering (e.g., 2 days “off”),followed by 5 consecutive days administering (e.g., 5 days “on”), per28-day cycle. In some embodiments, the administering may be performed,e.g., for 14 total days, wherein the 14 days of administering comprise 7days “on” followed by 7 days “off” followed by 7 days “on”, per 28-daycycle. In some embodiments, the administering may be performed, e.g.,for 9 total days, wherein the 9 days of administering comprise 3consecutive days “on” followed by 4 consecutive days “off”, followed by3 consecutive days “on” followed by 4 consecutive days “off”, followedby 3 consecutive days “on”, per 28-day cycle. In some embodiments, theadministering may be performed, e.g., for 9 total days, wherein the 9days of administering comprise 3 non-consecutive days “on” (e.g., MWF),followed by 1 day “off”, followed by 3 non-consecutive days “on” (e.g.,MWF), followed by 1 day “off”, followed by 3 non-consecutive days “on”,per 28-day cycle.

In some embodiments, the administering steps (i) and (ii) may beperformed for one or more weeks per 28-day cycle, e.g., one week, twoweeks, three weeks, or four weeks per 28-day cycle. The weeks may beconsecutive and/or non-consecutive.

The administration regimen may include pretreatment and/orco-administration with at least one additional therapeutic agent. Insuch case, the compounds or composition of the invention, CDA substratedrug and/or at least one additional therapeutic agent may beadministered simultaneously, separately, or sequentially.

Examples of a chemotherapeutic agent include without limitation:alkylating agents (e.g., which may include doxorubicin,cyclophosphamide, estramustine, carmustine, mitomycin, bleomycin and thelike); antimetabolites (e.g., which may include 5-Fluoro-Uracil,capecitabine, gemcitabine, nelarabine, fludarabine, methotrexate and thelike); platinating agents (e.g., which may include cisplatin,oxaliplatin, carboplatin and the like); topoisomerase inhibitors (e.g.,which may include topotecan, irinotecan, etoposide and the like);tubulin agents (e.g., which may include paclitaxel, docetaxel,vinorelbine, vinblastine, vincristine, other taxanes, epothilones, andthe like); signaling inhibitors (e.g., kinase inhibitors, antibodies,farnesyltransferase inhibitors, and the like); and otherchemotherapeutic agents (e.g., tamoxifen, anti-mitotic agents such aspolo-like kinase inhibitors or aurora kinase inhibitors, and the like).

Examples of administration regimens include, without limitation:administration of each compound, composition, CDA substrate drug, and/ortherapeutic agent in a sequential manner; and co-administration of eachcompound, composition, CDA substrate drug, and/or therapeutic agent in asubstantially simultaneous manner (e.g., as in a single unit dosageform) or in multiple, separate unit dosage forms for each compound,composition, CDA substrate drug, and/or therapeutic agent.

It will be appreciated by those skilled in the art that the “effectiveamount” or “dose level” will depend on various factors such as theparticular administration mode, administration regimen, compound, andcomposition selected, and the particular disease and patient beingtreated. For example, the appropriate dose level may vary depending uponthe activity, rate of excretion and possible toxicity of the specificcompound or composition employed; the age, body weight, general health,gender and diet of the patient being treated; the frequency ofadministration; the other therapeutic agent(s) being co-administered;and the type and severity of the disease.

The present invention contemplates per dose amounts on the order ofabout 5 mg to about 35 mg per dose of decitabine or the equivalentamount of other CDA substrate drugs. As used herein, the term“equivalent amount” refers to any dose amount that results in equivalentefficacy, such as equivalent epigenetic effects (e.g., LINE-1demethylation, e.g., % F cell expansion) and/or equivalent reduction ofmyelosuppressive effects (e.g., neutropenia).

In some embodiments, the dose of decitabine may be about 5, 10, 15, 20,25, 30, or 35 mg per dose, or any value or range therein. In someembodiments, the dose of decitabine may be less than about 35, 30, 25,20, 15, or 10 mg. The present invention further contemplates cumulativedose amounts on the order of about 15 mg to about 150 mg cumulative per28-day cycle of treatment of decitabine or the equivalent amount ofother CDA substrate drugs (e.g., about 10 mg to about 450 mg5-azacytidine). In some embodiments, the cumulative dose of decitabineper 28-day cycle may be, e.g., about 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 146, 147, 148, 149, or 150 mg or any value orrange therein. For example, in some embodiments, the amount ofdecitabine per dose may be about 5 mg to about 35 mg, about 5 mg toabout 30 mg, about 5 mg to about 25 mg, about 5 mg to about 20 mg, about5 mg to about 15 mg, about 5 mg to about 10 mg, about 7 mg to about 20mg, or about 5 mg, about 10 mg, about 15 mg, or about 35 mg. In someembodiments, the cumulative dose of decitabine per 28-day cycle may be,for example, about 15 mg to about 150 mg, about 15 mg to about 125 mg,about 15 mg to about 100 mg, about 15 mg to about 75 mg, about 15 mg toabout 50 mg, about 15 mg to about 40 mg, about 15 mg to about 30 mg,about 30 mg to about 120 mg, about 45 mg to about 125 mg, about 50 mg toabout 150 mg, about 65 mg to about 125 mg, or about 75 mg to about 140mg. In some embodiments, the cumulative dose of decitabine per 28-daycycle may be, e.g., about 50 mg, or about 75 mg, or about 130 mg. Doselevels, mode of administration, and administration regimen may bemodified by those skilled in the art using known techniques as judgednecessary for the subject (e.g., the patient).

In some embodiments, the dose of 5-azacytidine may be about 10, 15, 20,25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 430, 435, 440, 445, or 450 mg per dose, or anyvalue or range therein. In some embodiments, the dose of 5-azacytidinemay be less than about 450, 445, 440, 435, 430, 425, 400, 375, 350, 325,300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, 45, 40, 35, 30, 25,20, 15, or 10 mg. The present invention further contemplates cumulativedose amounts on the order of about 15 mg to about 150 mg cumulative per28-day cycle of treatment of decitabine or the equivalent amount ofother CDA substrate drugs. In some embodiments, the cumulative dose of5-azacytidine per 28-day cycle may be, e.g., about 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300,2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400 or 3500mg or any value or range therein. For example, in some embodiments, theamount of 5-azacytidine per dose may be about 10 mg to about 35 mg,about 35 mg to about 400 mg, about 10 mg to about 450 mg, about 15 mg toabout 420 mg, about 20 mg to about 350 mg, about 35 mg to about 300 mg,about 12 mg to about 150 mg, or about 25 mg, or about 250 mg. In someembodiments, the cumulative dose of 5-azacytidine per 28-day cycle maybe, for example, about 10 mg to about 3500 mg, about 15 mg to about 3125mg, about 15 mg to about 2940 mg, about 10 mg to about 1400 mg, about200 mg to about 3500 mg, or about 50 mg to about 2750 mg. In someembodiments, the cumulative dose of 5-azacytidine per 28-day cycle maybe, e.g., about 10 mg, about 200 mg, about 1400, about 2940, or about3150 mg. Dose levels, mode of administration, and administration regimenmay be modified by those skilled in the art using known techniques asjudged necessary for the subject (e.g., the patient).

The present invention contemplates per dose amounts on the order ofabout 40 mg to about 1000 mg per dose of cedazuridine. In someembodiments, the dose may be about 75 mg, 100 mg, 150 mg, 200 mg, 250mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700mg, 750 mg, 800 mg, 850 mg, 900 mg, 925 mg, 950 mg, 975 mg or 1000 mgper dose, or any value or range therein. For example, in someembodiments, the dose amount may be about 100 mg per dose ofcedazuridine. In some embodiments, the dose amount may be less thanabout 100 mg of cedazuridine. The present invention further contemplatescumulative dose amounts on the order of about 40 mg to about 7000 mgcumulative per 28-day cycle of treatment of cedazuridine. In someembodiments, the cumulative dose per 28-day cycle may be, e.g., about40, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800,2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000,4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200,5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400,6500, 6600, 6700, 6800, 6900, 6925, 6950, 6975, or 7000 mg or any valueor range therein. For example, in some embodiments, the cumulative doseof cedazuridine per 28-day cycle may be, for example, about 40 mg toabout 150 mg, about 65 mg to about 3500 mg, about 75 mg to about 6500mg, about 40 mg to about 500 mg, about 100 mg to about 250 mg, or about75 mg to about 1000 mg. In some embodiments, the cumulative dose ofcedazuridine per 28-day cycle may be, e.g., about 100 mg, about 1000 mg,about 725 mg, about 2000 mg, about 6500 mg, about 3500 mg, about 2500mg, about 4750 mg, or about 475 mg. Dose levels, mode of administration,and administration regimen may be modified by those skilled in the artusing known techniques as judged necessary for the subject (e.g., thepatient).

Formulations and Kits

The pharmaceutical compositions (e.g., compound, composition, CDAsubstrate drug, and/or therapeutic agent) of the invention may beformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (for example, aqueous or non-aqueous solutions or suspensions),tablets (for example, those targeted for buccal, sublingual and systemicabsorption), caplets, boluses, powders, granules, pastes for applicationto the tongue, hard gelatin capsules, soft gelatin capsules, mouthsprays, troches, lozenges, pellets, syrups, suspensions, elixirs,liquids, emulsions and microemulsions; (2) parenteral administration,for example, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension; (3) topicalapplication, for example, as a cream, ointment, patch, pad or sprayapplied to the skin; (4) intravaginally or intrarectally, for example,as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7)transdermally; or (8) nasally. The pharmaceutical compositions may beformulated for immediate, sustained or controlled release.

In some embodiments, the pharmaceutical compositions are formulated fororal administration. In further embodiments, the pharmaceuticalcompositions are formulated for oral administration in solid form.

Pharmaceutical compositions of the invention can be prepared using knownmaterials and techniques, which may include but are not limited tomixing and/or blending the compound of the invention with thepharmaceutically acceptable excipient and optional therapeutic agent(s).

Another aspect of the present invention relates to a unit dosage formand a kit comprising at least one unit dosage form, which unit dosageform comprises a compound or pharmaceutical composition of theinvention. In some embodiments, the unit dosage form comprises about 5,10, 15, 25, 20, 30, or 35 mg decitabine or an equivalent amount ofanother CDA substrate drug (e.g., about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 430, 435, 440, 445, or 450 mg 5-azacytidine). In some embodiments,the unit dosage form of decitabine may comprise less than about 35, 30,25, 20, 15, or 10 mg. For example, in some embodiments, the unit dosageform of decitabine may be about 5 mg to about 35 mg, about 5 mg to about30 mg, about 5 mg to about 25 mg, about 5 mg to about 20 mg, about 5 mgto about 15 mg, about 5 mg to about 10 mg, about 7 mg to about 20 mg, orabout 5 mg, about 10 mg, about 15 mg, or about 35 mg. In someembodiments, the unit dosage form of 5-azacytidine may be about 10 mg toabout 35 mg, about 35 mg to about 400 mg, about 10 mg to about 450 mg,about 15 mg to about 420 mg, about 20 mg to about 350 mg, about 35 mg toabout 300 mg, about 12 mg to about 150 mg, or about 25 mg, or about 250mg. In some embodiments, the unit dosage form of cedazuridine comprisesabout 40 mg to about 1000 mg cedazuridine. For example, in someembodiments, the unit dosage form of cedazuridine may comprise about 40mg, about 75 mg, about 100 mg, about 200 mg, about 100 mg, about 750 mg,about 500 mg, or about 250 mg cedazuridine. In some embodiments, theunit dosage form of cedazuridine may be less than about 100 mg. In someembodiments, the unit dosage form comprises a CDA substrate drug (e.g.,decitabine, e.g., 5-azacytidine) in the amount described above andcedazuridine in the amount described above.

The kit may further comprise a container and/or a package suitable forcommercial sale. The container can be in any conventional shape or formas known in the art which is made of a pharmaceutically acceptablematerial, such as a paper or cardboard box, a glass or plastic bottle orjar, a re-sealable bag, or a blister pack with individual dosages forpressing out of the pack according to a therapeutic schedule. More thanone container can be used together in a single package. For example,tablets may be contained in a blister pack which is in turn containedwithin a box.

The kit may further comprise information. The information may beprovided on a readable medium. The readable medium may comprise a label.The information may be directed towards a physician, pharmacist orpatient. The information may indicate that the unit dosage form maycause one or more adverse effects. The information may compriseinstructions for administering the unit dosage form, such as in a mannerdescribed herein. These instructions may be provided in a variety ofways. For example, the information may include a table including avariety of weights or weight ranges and appropriate dosages for eachweight or weight range.

The information can be associated with the container, for example, bybeing: written on a label (e.g., the prescription label or a separatelabel) adhesively affixed to a container; included inside a container asa written package insert; applied directly to the container such asbeing printed on the wall of a box or blister pack; or attached as bybeing tied or taped, for example as an instructional card affixed to theneck of a bottle via a string, cord or other line, lanyard or tethertype device.

It will be apparent to those skilled in the art that specificembodiments of the present invention may be directed to one, some or allof the above-indicated aspects as well as other aspects, and mayencompass one, some or all of the above- and below-indicatedembodiments, as well as other embodiments.

Other than in the working examples, or where otherwise indicated, allnumbers expressing quantities of ingredients, reaction conditions, andso forth used in the specification and claims are to be understood asbeing modified by the term “about”. Accordingly, unless indicated to thecontrary, such numbers are approximations that may vary depending uponthe desired properties sought to be obtained by the present invention.At the very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should be construed in light of the number of significantdigits and ordinary rounding techniques.

While the numerical ranges and parameters setting forth the broad scopeof the invention are approximations, the numerical values set forth inthe working examples are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in their respective testingmeasurements.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

EXAMPLES Example 1: Evaluation of Low Dose CombinationDecitabine/Cedazuridine Systems Pharmacology Model

Decitabine (DAC) is a well characterized hypomethylating agent (HMA)which is incorporated into DNA during the S-phase of cell cycle,inhibiting methylation of antitumor genes and inducing G2/M arrest.Decitabine is approved for treatment of intermediate to high risk MDS,but is rapidly degraded by cytidine deaminase (CDA), resulting in poororal decitabine bioavailability and systemic exposures. ASTX727 as afixed dose oral combination of the CDA inhibitor E7727 and decitabinehas been tested for the treatment of patients with intermediate- andhigh-risk MDS or chronic myelomonocytic leukemia (CMML). Low risk MDS(e.g., IPSS low or IPSS Intermediate-1) patients present with less than5% leukemic blasts in bone marrow and anemia or cytopenia. As treatmentwith decitabine causes side effects such as neutropenia and/orthrombocytopenia, the objective of this study was to use quantitativesystems pharmacology (QSP) modeling to describe the effect of decitabineon neutrophils and simulate regimens to minimize myelosuppression.

A QSP model was previously developed describing myeloblasts cell cycle,leukemic blasts, neutrophils and platelets in physiological compartments(e.g., bone marrow and blood), and PK of decitabine after i.v. infusion,and was applied to model dosing with subcutaneous guadecitabine(SGI-110, a dinucleotide of decitabine linked to deoxyguanosine; 2015ASCPT: Systems Pharmacology Modeling for Hypomethylating agentsDecitabine and SGI-110 for Evaluation of AML treatment by targeting theS-phase with prolonged Pharmacokinetic exposures, Oganesian et al., 0)and to model oral ASTX727 (2017 ASCPT: Development of a Semi-MechanisticPK/PD Model of an Oral Fixed Dose Combination (FDC) of CytidineDeaminase Inhibitor E7727 with Decitabine (ASTX727) in Subjects withMyelodysplastic Syndromes, Burroughs et al.). The present modeldescribes myeloblasts cell cycle, leukemic blasts, neutrophils andplatelets in physiological compartments (e.g., bone marrow and blood),PK of decitabine, and PD marker of LINE-1 demethylation changesfollowing treatment with HMAs, as well as effects of decitabine onleukemic cells, neutrophils and platelets to model various low-dosecombinations of decitabine/cedazuridine.

Key differential equations for the model include those shown in FIG. 2.

For this study, the model was successfully calibrated and validatedagainst various types of data and successfully reproduced clinical dataon neutrophil counts following treatment with guadecitabine in AMLpatients (FIG. 1). Thus, the model showed that decitabine-inducedneutropenia is not the result of gene demethylation or decitabineincorporation into DNA, but has a complex mechanism that depends ondecitabine levels in plasma.

Simulations with several regimens using low doses of decitabine forASTX727 administration were performed. The model predicted thatneutrophil levels depend on the dose and frequency of ASTX727administration (FIGS. 3A-3B). The model further suggested that the leasttoxic ASTX-LD regimen among those simulated was the 5 mg DAC/100 mgE7727 daily (e.g., “ 5/100 mg”) for 5 days for 2 weeks, repeated every28-day cycle. ASTX727-LD regimens of 10/100 mg daily for 3 days for 3weeks, and 20/100 mg daily for 3 days for 2 weeks had a similar effecton neutrophil dynamics, whereas the effect on leukemic blasts was betterwith the 10/100 mg daily for 3 days for 3 weeks regimen.

Thus, the updated model adequately described available data onneutrophil dynamics after treatment with guadecitabine, and allowedsimulation of lower doses of varying regimens for optimization oftreatment with ASTX727 to minimize decitabine-mediated adverse effectssuch as neutropenia and potentially maximize efficacy. The modelsuggested that the optimal regimen of ASTX727 that induces minimalchanges in neutrophil counts and with potential maximal effects onleukemic blasts in the bone marrow modeled was a low dose of 5 mgdecitabine with 100 mg E7727, dosed daily for 5 days for 2 weeks ofevery 28-day cycle.

Example 2: Low Dose Combination Decitabine/Cedazuridine with ExtendedSchedule in Subjects with Lower Risk MDS

A Phase 1-2, multicenter, open-label study of various combinationdecitabine/cedazuridine (referred to as ASTX727) low dose (LD) doses andschedules was performed to assess the safety, pharmacodynamics,pharmacokinetics, and hematologic response in subjects with anInternational Prognostic Scoring System (IPSS) risk category of low-riskor Intermediate-1 (Int-1) MDS.

Inclusion criteria included men or women 18 or older with IPSS low riskor Int-1 MDS, an Eastern Cooperative Oncology Group (ECOG) status of 0to 2, adequate hepatic and renal function, as well as at least either ared blood cell transfusion dependence of 2 or more units of RBCs orhemoglobin of less than 8.5 g/dL at baseline, an ANC of less than0.5×10⁹/L prior to enrollment, or platelet counts of less than 50×10⁹/Lprior to enrollment.

Exclusion criteria included prior treatment of other investigationaldrugs or therapy within 2 weeks before study, or 5 half-lives, whicheverlonger; prior treatment of MDS concluded less than 1 month prior tostudy; diagnosis of CMML; uncontrolled systemic diseases or activeuncontrolled infections, known mental illness or substanceabuse/addiction; life-threatening illness; active infection of HIV orhepatitis; or other prior malignancy unless the subject has been diseasefree for at least 1 year.

Randomization was stratified by diagnostic category (low risk vs. Int-1IPSS), baseline ANC (≤10⁹/L vs. ≥10⁹/L), and ECOG Performance Score (0-1vs. 2). Phase 1 Stage A subjects were randomized in a 1:1:1 ratio intothree cohorts of 6 subjects, each in a 10-day schedule in a 28-daycycle, as shown in Table 1.

TABLE 1 Phase 1 Stage A Cohorts (10-day Schedule) Total Total CohortDecitabine Cedazuridine Schedule Cedazuridine Decitabine (n = 6 DoseDose (28-Day Dose Dose each) (mg) (mg) Cycle) (mg/cycle) (mg/cycle) 1 5100 Daily × 5, 1000 50 2 10 Off × 2, 100 3 15 Daily × 5 150

An additional cohort of 15 mg decitabine and 100 mg cedazuridine wasalso contemplated. The goal of this study was to find a dose andschedule of ASTX727 LD which provides active epigenetic and clinicaleffects with minimum myelosuppression. All daily doses were lower thanpreviously tested. The cumulative doses/cycle of decitabine were lessthan the approved regimen. The cumulative dose/cycle of cedazuridine wasincreased in Stage 1 and 2 of dose escalation over previously testeddoses. Patient read-outs included % LINE-1 methylation change frombaseline, area under the curve (AUC), maximum plasma concentration(Cmax), time to reach maximum concentration (Tmax), half-life (t½),hematologic response, treatment time required for bone marrow blasts toexpand by ≥50% and/or to greater than 5% of total sample cells, as wellas leukemia-free survival and overall survival.

The 5 mg cohort showed 2 patients that experienced neutropenia ofgreater than two weeks or to a level leading to dose interruption. The10 mg cohort showed three patients with neutropenia of greater than twoweeks. Hemoglobin levels, platelet counts, and ANC of the 5 mg cohortand 10 mg cohort were analyzed.

Treatment showed LINE-1 demethylation as well as hemoglobin-F carryingcell expansion (“F cells”) in both the 5 mg and 10 mg cohorts,indicators of epigenetic effects. An F cell percentage of greater than20% is also used as a correlative with clinical effect in the sicklecell field. Mean decitabine concentrations in plasma are shown in FIG.4. Decitabine concentrations were detectable in plasma up to four to sixhours for the 5 and 10 mg cohorts. Mean PK parameters are shown in Table2. There was an approximate 2-fold increase in exposures from day 1 today 5, and from 5 mg to 10 mg. Comparisons of total cycle area under thecurve (AUC) are shown in Table 3.

TABLE 2 PK Parameters. C_(max) ¹ T_(max) ² AUC_(0-24 h) ¹ Occasion N(ng/mL) (hr) (ng*hr/mL) Cohort 1 C1D1 10  6.1 (76) 0.5 (0.25-1.5)  6.3(70) (5 mg) C1D5 10 12.6 (85) 0.5 (0.25-1.5) 12.1 (64) Cohort 2 C1D1 412.2 (66) 1.0 (0.5-2.0)  13.2 (36) (10 mg) C1D5 3 31.0 (80) 0.5(0.25-0.5) 26.3 (64)

TABLE 3 Total Cycle AUC Comparison. Daily decitabine Cycle cumulative %FDC cycle Total cycle dose dose cumulative AUC_(0-24 h) % FDC totalCohort (mg) (mg) dose (ng*hr/mL) cycle AUC_(0-24 h) 1 5 50 29% 115.2 14%2 10 100 57% 249.9 30% Stan- 35 175 100%  823.1 100%  dard dose¹

The LINE-1 demethylation and hemoglobin F (HbF) induction resultsindicate that the doses tested exerted epigenetic effects in adose-responsive manner. Hypomethylation of LINE-1 observed in the 10 mgcohort showed higher magnitude achieved by higher risk MDS patientsgiven higher doses in early phase studies.

Example 3: Additional Schedules for Low Dose CombinationDecitabine/Cedazuridine with Extended Schedule in Subjects with LowerRisk MDS

Patients are enrolled in cohorts of 5 mg or 10 mg of varying schedulesper 28-day cycle, as shown in Tables 4 and 5.

TABLE 4 Cohorts on 3-day or 5-day treatment schedules. Total TotalCohort Decitabine Cedazuridine Schedule Cedazuridine Decitabine N = 6Dose Dose (28-day Dose Dose each (mg) (mg) cycle) (mg/cycle) (mg/cycle)1 5 100 MWF 600 30 1 w on/1 w off 2 10 MWF 600 60 1 w on/1 w off 3 5 MWF900 45 3 w on/1 w off 4 10 MWF 900 90 3 w on/1 w off 5 5 Daily × 5 50025 6 10 Daily × 5 500 50

TABLE 5 Cohorts on 7-day or 14-day treatment schedules. Total TotalCohort Decitabine Cedazuridine Schedule Cedazuridine Decitabine (n = 6Dose Dose (28-Day Dose Dose each) (mg) (mg) Cycle) (mg/cycle) (mg/cycle)1 5 100 Daily × 7, 1400 70 2 10 Off × 7, 140 Daily × 7, Off × 7 3 5Daily × 70 4 10 14, 140 Off × 14

Additional schedules include a cohort receiving 20 mg of decitabine and100 mg of cedazuridine for 5 days (e.g., 5 consecutive days) per 28-daycycle, a cohort receiving 10 mg of decitabine and 100 mg of cedazuridinefor 7 days (e.g., 7 consecutive days) per 28-day cycle, and a cohortreceiving 20 mg decitabine for 5 days (e.g., 5 consecutive days) per28-day cycle, as well as a control group receiving 35 mg decitabine and100 mg of cedazuridine for 3 days per 28-day cycle. Schedule changes indosing amount (e.g., between 5 mg to 35 mg decitabine per dose),cumulative dosing, and administration (e.g., between 3 days to 21 daysper 28-day cycle, given on consecutive days and/or non-consecutive days)are contemplated as well.

Dose-limiting toxicity is assessed to eliminate cohorts that experiencethe following events: (1) drug-related≥Grade 3 non-hematologic events(except nausea, vomiting, or diarrhea well controlled by symptomatictreatment); (2) hematologic events (e.g., Grade 4 anemia, platelet, orANC counts) not existing at baseline and lasting greater than 2 weeks induration; and (3) drug-related toxicity causing delay of greater than 2weeks after Cycle 1 to initiation of next scheduled cycle.

Example 4: Combination Decitabine/Cedazuridine Oral Vs. IV Delivery

Table 6 shows tabulated results of standard schedule treatment of oralcombination decitabine/cedazuridine (ASTX727) resulted in greater levelsof drug availability (by % AUC) in patients over those receivingdecitabine and cedazuridine (E7727) intravenously.

TABLE 6 % of AUC Co- ASTX727 Oral Dose (mg) 5-Days total (AUC_(last))(Oral/ hort DAC¹ E7727 N Oral IV IV) 1 20 40  5² 260 753 35% 2 20 60 6346 899 39% 3 20 100 6 482 992 49% 4 40 100 6 1120 775 144%  5 30 10019³ 701 852 85% ¹DAC oral dose not adjusted by weight or BSA; ²Onesignificant outlier excluded from PK analysis; ³Includes 13 patient doseexpansion.

Cohort 4 oral delivered decitabine (DAC) at 40 mg exposure given withoral cedazuridine achieved and exceeded AUC of i.v. delivered decitabineat 20 mg/m² at an oral/i.v. ratio of 144%. Cohort 5 oral delivereddecitabine at 30 mg given with oral cedazuridine exposure was 85% ofi.v. delivered decitabine at 20 mg/m². Oral delivered decitabine dose of35 mg in ASTX727 are expected to achieve an AUC of about 85% to about140% of i.v. delivered decitabine. FIG. 5 shows % demethylation ofLINE-1 achieved by the cohorts of Table 6. Cohorts 4 and 5 achievedLINE-1 demethylation from baseline of greater than 10%, comparable tohistorical data of i.v. delivered decitabine.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. Although the invention hasbeen described in detail with reference to preferred embodiments,variations and modifications exist within the scope and spirit of theinvention as described and defined in the following claims.

What is claimed is:
 1. A method for treating cancer in a subject in needthereof, wherein said cancer is myelodysplastic syndromes (MDS),leukemia, or lymphoma, comprising administering to the subject: (i)decitabine at a dose of about 5 mg to about 15 mg; and (ii) an effectiveamount of cedazuridine, thereby treating cancer in the subject.
 2. Themethod of claim 1, wherein the leukemia is acute lymphocytic leukemia(ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML),myeloproliferative neoplasms (MPN), or chronic myelomonocytic leukemia(CMML).
 3. The method of claim 1, wherein the MDS is InternationalPrognostic Scoring System (IPSS) low and/or IPSS intermediate 1 MDS. 4.The method of claim 1, wherein the administering is performed in onecomposition.
 5. The method of claim 1, wherein the decitabine isadministered at a cumulative dose of from about 15 mg to about 75 mgcumulative per 28-day cycle of treatment.
 6. The method of claim 1,wherein the effective amount of cedazuridine is from about 40 mg perdose to about 1000 mg per dose.
 7. The method of claim 1, wherein theeffective amount of cedazuridine is from about 100 mg to about 7000 mgcumulative per 28-day cycle of treatment.
 8. The method of claim 1,wherein the administering reduces absolute neutrophil count (ANC) in thesubject to less than 0.5×10⁹ cells/L for no more than two weeksfollowing a 28-day cycle of treatment.
 9. The method of claim 1, whereinthe administering expands hemoglobin F-expressing.
 10. The method ofclaim 1, wherein the administering reduces DNA methylation in thesubject by at least 5% as compared to DNA methylation in the subjectprior to the administering.